Development of compost maturity and Actinobacteria populations during full-scale composting of organic household waste.

AIMS: This study investigates changes in microbiological and physicochemical parameters during large-scale, thermophilic composting of a single batch of municipal organic waste. The inter-relationships between the microbial biomass and community structure as well as several physicochemical parameters and estimates of maturation were evaluated. METHODS AND RESULTS: Analyses of signature fatty acids with the phospholipid fatty acid and ester-linked methods showed that the total microbial biomass was highest during the early thermophilic phase. The contribution of signature 10Me fatty acids from Actinobacteria indicated a relatively constant proportion around 10% of the microbial community. However, analyses of the Actinobacteria species composition with a PCR-denaturing gradient gel electrophoresis approach targeting 16S rRNA genes demonstrated clear shifts in the community structure. CONCLUSIONS: This study demonstrates that compost quality, particularly maturity, is linked to the composition of the microbial community structure, but further studies in other full-scale systems are needed to validate the generality of these findings. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of signature lipid and nucleic acid-based analyses greatly expands the specificity and the scope for assessing the microbial community composition in composts. The results presented in this study give new information on how the development of the compost microbial community is connected to curing and maturation in the later stages of composting, and emphasizes the role of Actinobacteria in this respect.

Flow cytometry and allele-specific oligonucleotide PCR are equally effective in detection of minimal residual disease in ALL.

The analysis of minimal residual disease (MRD) has assumed a growing role in the follow-up of patients with acute lymphoblastic leukemia (ALL). We have applied multiparameter flow cytometry (FC) with 'live-gate' analysis and allele-specific oligonucleotide (ASO)-PCR detecting leukemia-specific T cell receptor gamma and delta gene rearrangements for MRD follow-up in 30 ALL patients. The comparison of results obtained in 89 follow-up samples from 23 patients showed significantly consistent results in 70 samples (78%); (P < 0.001). Bone marrow samples taken during the first phase of treatment (during or immediately after induction) showed a lower level of consistency when compared to samples taken during later phases of treatment (69% vs 85% consistent results, respectively). Some of the discrepant results were due to low cellularity of the samples obtained for FC and some due to the presence of PCR inhibitors. Of 29 patients evaluated at the end of the induction treatment, 18 (62%) had detectable levels of MRD and six of these patients suffered relapse. In all these patients MRD levels by FC increased preceding relapse. Our results suggest that FC offers a MRD detection tool that can be easily applied in clinical practice and is as informative as molecular methods.

Interleukin-10 mRNA expression in B-cell chronic lymphocytic leukaemia inversely correlates with progression of disease.

Interleukin-10 (IL-10) has been shown in vitro to inhibit survival and spontaneous DNA synthesis in B-cell chronic lymphocytic leukaemia (B-CELL) cells by induction of programmed cell death. We have analysed the presence of mRNA transcripts for IL-10 in purified B-CLL cells from 35 patients by RT-PCR. Transcripts for IL-10 were detected in 11/20 patients with non-progressive disease. In cell preparations from patients with progressive B-CLL IL-10 mRNA were detected in only 2/15 samples (P < or = 0.01). The Epstein-Barr virus status of the cells did not account for the difference in IL-10 mRNA expression observed between the two groups of patients. Thus, IL-10 mRNA expression in leukaemic cells from patients with B-CLL was strongly associated with non-progressive disease. This finding may support other observations suggesting that IL-10 might be a candidate for immune therapy of progressive B-CLL.

Cloning of the rat and human prostaglandin F2 alpha receptors and the expression of the rat prostaglandin F2 alpha receptor.

We have cloned the FP receptor from rat corpus luteum and human uterus cDNA libraries, respectively. The coding DNA sequence in the rat cDNA is 1101 bp and is similar to the mouse cDNA coding for a receptor protein of 366 amino acids. The human sequence shows a 5 bp deficiency in the 3' region, truncating the coding sequence to 359 amino acids. Northern blot analysis indicates highest expression in the ovary. Cell lines have been established giving stable expression of the FP receptor. Activation of the cloned FP receptor gave an increase in intracellular calcium, indicating signaling via phospholipase C-mediated phosphoinositide turnover. Using [3H]PGF2 alpha, binding of PGs showed the rank order of fluprostenol > PhXA70 > PGF2 alpha > or = PhXA85 > PGD2 > PGE2.

The role of IgA determination by ELISA in the early serodiagnosis of Mycoplasma pneumoniae infection, in relation to IgG and mu-capture IgM methods.

Enzyme-linked immunosorbent assay (ELISA) for IgA, IgG and IgM was evaluated with sera from 50 adult patients with pneumonia, selected on the basis of a positive complement fixation (CF) test for diagnosis of Mycoplasma pneumoniae infection and with sera from 105 healthy blood donors. The ELISA antigen for IgG and IgA was a sonicated suspension of M. pneumoniae solubilised by deoxycholate. For the IgM assay, the same antigen was directly conjugated to alkaline phosphatase and used in a mu-capture format. ELISA gave positive results with high or rising titres for one or several antibody classes in 47 (94%) patients. In two of the three ELISA-negative cases, the diagnosis of M. pneumoniae infection indicated by the CF test seemed unlikely on clinical grounds. Specific IgA antibodies was developed more regularly and more rapidly than IgM. IgA titres also started to decrease earlier than IgM or the late-peaking IgG response. Thus, the determination of IgA antibodies was found to be valuable for the early diagnosis of M. pneumoniae infection. The study also demonstrated that the determination of all three antibody classes is necessary to obtain an optimal level of serodiagnosis.

Substance P-, calcitonin gene-related peptide, growth-associated protein-43, and neurotrophin receptor-like immunoreactivity associated with unmyelinated axons in feline ventral roots and pia mater.

The spinal pia mater receives a rich innervation of small sensory axons via the ventral roots. Other sensory axons enter the ventral roots but end blindly or turn abruptly in hairpin loop-like formations and continue in a distal direction. In the present study, the content of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, growth-associated protein (GAP-43)-, and low-affinity neurotrophin receptor protein (p75NGFr)-like immunoreactivity (-LI) associated with these different types of sensory axons was assessed with light and electron microscopic immunohistochemical techniques. In addition, the binding of antibodies against synthetic peptides representing unique sequences of residues in the products of the trk and trkB protooncogenes was analyzed. These genes encode membrane spanning proteins, which have been shown to constitute specific high affinity binding sites for several members of the nerve growth factor family of neurotrophic factors. The results of the present study imply that the ventral root afferents comprise several different types of sensory axons, which all contain SP-, CGRP-, GAP-43-, and p75NGFr-like immunoreactivities. In addition, at least some of the presumed sensory fiber bundles in ventral roots and the pia mater were immunoreactive for the trkB gene product. Moreover, leptomeningeal cells and nonneuronal cells of the ventral roots were shown to bind antibodies to both the trk and trkB gene products. The ventral root afferents seem to share their immunohistochemical pattern with pain-transducing axons at some other locations, such as the tooth pulp. The contents of SP- and CGRP-LI in sensory axons that reach the central nervous system (CNS) through the ventral root indicate that ventral root afferents may be involved in sensory mechanisms, such as the ventral root pain reaction, as well as in the control of the pial blood vessels. The demonstration of GAP-43 and neurotrophin receptor-immunoreactivities associated with unmyelinated fibers in ventral roots and the pia mater is discussed in relation to previous reports on postnatal plasticity in these axonal populations.

Growth of ascending spinal axons in CNS scar tissue.

The aim of the present study was to test the capacity of spinal cord scar tissue to assist and sustain axon regrowth. In adult rats and cats the dorsal funiculus (DF) was cut at mid-thoracic or lumbar level, and a superficial incision in the DF rostral to the lesion was made in order to extend the penetrating lesion. Axonal tracing in rats 50-100 days postinjury with anterogradely transported wheatgerm agglutinin-conjugated horseradish peroxidase or rhodamine-conjugated dextran demonstrated that nerve fibers had entered the scar tissue. Axon ingrowth in the scar was further indicated by axonal immunoreactivity to the growth-associated protein GAP-43. The scar tissue showed low-affinity neurotropin receptor-like immunoreactivity in association with blood vessels and in the interstitium. The integrity of the blood-brain barrier in the extended dorsal funiculus lesion was disrupted for at least 11 months postinjury, assessed by i.v. injections of free HRP or Evans blue. The present study shows that penetrating injury in the dorsal funiculus produces a CNS environment permissive for axonal sprouting and that PNS influence is not necessary for spinal tract regrowth. A possible relationship between the absence of an intact BBB and injury-induced axonal sprouting is discussed.

Extravasation of staphylococcal alpha-toxin in normal and injured CNS regions lacking blood-brain barrier function: observations after ventral root replantation.

Staphylococcus aureus plays an important role as a bacterial pathogen after traumatic injury. The majority of isolated strains produces alpha-toxin, a 33-kDa protein, with membrane-damaging and lethal effects. The central nervous system (CNS) has been considered as the possible target for the lethal action of this toxin. A transfer of alpha-toxin across an intact blood-brain barrier (BBB) is however unlikely. The aim of the present study was to determine if alpha-toxin is accumulated in CNS regions which lack the BBB function. The distribution of alpha-toxin after intravascular injections, in normal mice and rats as well as in rats subjected to ventral root replantation, was assessed using immunogold technique. The results show that, although alpha-toxin does not cross the BBB, alpha-toxin-like immunoreactivity could be detected in the area postrema and at the optic nerve-retinal junction. Extravasation of alpha-toxin was also shown to occur in the spinal cord even 22 months after ventral root replantation. This finding suggests that axon regeneration after ventral root replantation takes place in a macromolecular environment which is totally different from the normal CNS. The implications of vascular spread of alpha-toxin to regions devoid of BBB function are discussed in relation to the bacterial infections which might complicate severe spinal injuries.

Regeneration after spinal nerve root injury.

Spinal nerve root avulsion has been considered as a central nervous type of injury and therefore not repaired surgically in man. The possibility for axonal regeneration after root avulsion or root lesion has been investigated in laboratory animals by means of up to date neurophysiological, morphological and tracing techniques. It is shown that, after ventral root avulsion and implantation into the spinal cord, alpha and probably also gamma motoneurons are able to regenerate within the spinal cord for a considerable distance before entering the implanted root and reinnervate previously denervated skeletal muscles. The regenerated neurons were found to respond to afferent activity with excitatory or inhibitory responses, and the regenerated axons could conduct action potentials that elicited muscle twitch responses. After dorsal root injury in the adult animal, regeneration into the spinal cord does not occur. However, regeneration of primary sensory neurons into appropriate locations of the spinal cord can be demonstrated in immature animals.

Cytotoxic effects on viable human leukemic cells by combinations of lymphokine activated killer cells and monoclonal antibodies.

Earlier studies with individually phenotyped monoclonal antibody combinations and complement or lymphokine activated killer (LAK) cells showed that many acute myeloid leukemic cells were resistant to these cytotoxic agents when used singly. Therefore, a combination of both agents was studied. When the leukemic target cells were submitted to killer cells activated with 100 or 800 IU of recombinant interleukin-2 (rIL-2), only averages of 6.0 and 16.7% of the targets were killed respectively. When the remaining, refractory cells were confronted with a cocktail of individually phenotyped monoclonal antibodies and complement, an additional significant cell kill was obtained, but it amounted to only between 7.4 and 5.5% (for LAK-100 and LAK-800, respectively). In contrast, of the target cells initially refractory to the same cocktail of monoclonal antibodies, all were cross-resistant both to LAK-cells activated with 100 and to those activated with 800 IU of rIL-2. This cross-resistance was caused neither by sub-optimal LAK-cell activation, nor by antibody blocking of hypothetical LAK-cell receptors, since pre-incubation with monoclonal antibodies without complement did not inhibit LAK-cell cytotoxicity. Although only partial cross-resistance was found in the present study, it still remains that only a minority of the tumor cells could be killed. A higher in-vitro cell kill should be attempted prior to clinical trials in order to avoid clinical effects resembling those of a partial surgical tumor resection.

Demonstration of cross-reactions between pneumococci and alpha-streptococci using gold-labelled mono- and polyclonal antibodies and electron microscopy.

Cross-reactions between alpha-streptococci and the pneumococcal C-polysaccharide (PnC) were investigated using electron microscopy and immunogold labelling of bacterial cells. Monoclonal antibodies against two different determinants of the PnC molecule were used, one directed against the chain sugar of the repeating unit 2-acetamido-4-amino-2,4,6-trideoxygalactose (Sug) and the other against the phosphorylcholine residue. Two different immunogold techniques were tested, either by direct labelling of the monoclonal antibody or by using an antimouse immunogold conjugate to demonstrate binding of the monoclonal antibodies. Antibodies against the "Sug" determinant reacted only with pneumococci, whereas antibodies against the phosphorylcholine determinant bound to cross-reacting streptococci as well as to pneumococci. These results indicate that the cross-reacting antigens of the alpha-streptococci contain phosphorylcholine residues, but that they are not identical to the C-polysaccharide molecule.

Resistance of some leukemic blasts to lysis by lymphokine activated killer (LAK) cells.

Peripheral blood mononuclear cells (PBMC) from healthy donors and AML patients in remission were stimulated with phytohemagglutinin (PHA) and recombinant interleukin-2 (IL-2). These stimulated cells (lymphokine activated killer (LAK) cells) showed increased DNA synthesis as measured by 3H-Thymidine uptake. A synergistic effect of PHA and IL-2 was found. LAK cells' ability to kill acute myeloid leukemia (AML) blasts was investigated by the 51Cr release assay. LAK cells showed a cytotoxicity (over 10% specific 51Cr release) against 9/12 leukemic blasts, even at effector/target (E/T) ratios as low as 5:1. However, on average only 22.2% (SD 11.8) and 36.5% (SD 12.5) 51Cr release were obtained in 4- and 18-hour cytotoxicity assays, respectively, at an E/T ratio of 20:1. Leukemic blasts in 3/12 AML cases and normal PBMC were entirely resistant to lysis, even at an E/T ratio of 80:1. Susceptibility to lysis was not correlated to peanut-agglutinin receptor expression. LAK cells were more cytotoxic towards the K-562 cell line (natural killer activity) than unstimulated PBMC.

A highly specific two-site ELISA for pneumococcal C-polysaccharide using monoclonal and affinity-purified polyclonal antibodies.

A two-site ELISA for the detection of pneumococcal C-polysaccharide (PnC) has been developed. A monoclonal antibody directed against the phosphorylcholine residue of the PnC was used as catcher and an affinity-purified polyclonal anti-PnC rabbit antiserum for detection. Polyclonal antibodies against the PnC as well as capsular antigens were obtained by immunizing rabbits with type 1 pneumococci. Antibodies against the phosphorylcholine determinant of PnC could be removed by affinity purification. Remaining antibodies reacted in an ELISA with type 1 capsular polysaccharide as well as with PnC. Only in the fraction with the highest antibody activity against PnC, phosphorylcholine exhibited a slight inhibitory action. It is concluded that the purified antibody preparation reacted with an antigenic determinant shared by the two polysaccharides, in all probability a determinant associated with 2-acetamido-4-amino-2,4,6-trideoxygalactose which is the only monosaccharide component in common between PnC and the type 1 capsular polysaccharide. By the use of this affinity-purified antibody preparation, reactions with alpha-streptococci, occurring with non-purified serum, were abolished. The sensitivity and specificity of the test was determined using capsulated and non-capsulated pneumococci and alpha-streptococci known to cross-react with unpurified serum against the pneumococcal C-polysaccharide.

Etiologic diagnosis of pneumonia by antigen detection: crossreactions between pneumococcal C-polysaccharide and oral microorganisms.

Crossreactions between bacteria occurring more or less frequently in the respiratory tract were investigated using an enzyme-linked immunosorbent assay (ELISA) developed for the detection of pneumococcal C-polysaccharide. A collection of 218 strains was investigated: 30 Streptococcus pneumoniae, 120 alpha-streptococci, and 68 strains representing other species. Strong crossreactions were observed with 36% of the alpha-streptococci and with two of 11 Staphylococcus aureus strains. The collection of alpha-streptococci consisted of 90 fresh clinical isolates and 30 stock strains. Almost all crossreactions of alpha-streptococci were found among the clinical isolates. Among the stock strains only one of four Streptococcus mitis strains was positive. Pneumococcal C-polysaccharide and phosphorylcholine inhibited the reactions in ELISA with monoclonal antibodies against pneumococcal C-polysaccharide, as well as with a polyclonal antiserum against pneumococcal C-polysaccharide. We suggest that the cross reactions between alpha-streptococci and pneumococci depend on the presence of phosphorylcholine as a common antigenic determinant. The crossreaction in the ELISA with some Staphylococcus aureus strains may be explained by the presence of protein A binding to the Fc portion of the antibodies. When the 10 alpha-streptococci that showed the strongest crossreactions and ten pneumococci representing different types were tested in different concentrations the absorbance values were lower for most alpha-streptococci compared with the pneumococci. This explains that false positive results with alpha-streptococci do not seem to constitute a practical problem in this ELISA developed for detection of pneumococcal C-polysaccharide in samples from patients with pneumonia.

A new coagglutination test for detecting pneumococcal C-polysaccharide.

A new coagglutination test (PnC-CoA) for detecting pneumococcal C-polysaccharide (PnC) was compared with a commercial kit for detecting capsular polysaccharide using sputum samples from 105 patients with pneumonia. The sensitivity obtained with PnC-CoA was 95.8% and with the commercial kit 83.3%; the specificity was 96.5% and 91.2%, respectively. The PnC-CoA is simple to perform and it is a rapid, sensitive and specific test for detecting Streptococcus pneumoniae in sputa from adult patients with pneumonia.

Decrease in helper (T4+) lymphocytes following cimetidine treatment for duodenal ulcer.

We studied the possible in vivo influence of cimetidine on peripheral blood lymphocyte (PBL) subpopulations defined with monoclonal antibodies (MoAb) in eight haematologically normal patients with uncomplicated duodenal ulcers before cimetidine treatment, 1 week after the start, and finally 1 week following cessation of therapy. After cimetidine had been given for 3-5 weeks there was a significant decrease, compared with pretreatment numbers, in the proportion of T3+ cells (64.6 +/- 8.1 (mean +/- s.d.) v 51.0 +/- 7.8%) and in T4+ cells (47.3 +/- 4.3 v 30.8 +/- 4.7%). The number of T8+ cells was not affected (20.3 +/- 4.3 v 20.1 +/- 6.7%). These changes resulted in a significant reduction in the T4/T8 ratio (2.46 +/- 0.8 v 1.67 +/- 0.6). The total numbers of lymphocytes and monocytes as well as the percentage of B1+ lymphocytes did not change significantly. The observed decrease in T4/T8 ratio after cimetidine treatment is explained by a reduction in the number of T4+ cells and the appearance of a new subpopulation of T3-, T4-, T8-, B1-lymphocytes. The underlying mechanism, however, is not clear. Cimetidine does not seem to have a direct receptor-modulating effect, since in vitro exposure of normal lymphocytes to the drug did not change the proportions of the T cell subsets.

Effects of retinoids on in vitro differentiation of bone marrow cells in the myelodysplastic syndrome.

The effect of retinoids on cell differentiation in the myelodysplastic syndrome was studied in short-term liquid cultures of bone marrow from 13 patients. After incubation with 13-cis-retinoic acid there was a significant decrease in the percentages of promyelocytes and in Leu-M3-binding cells. Lue-M3 is mainly a monocyte marker, and retinoids thus seem to induce a shift from monocytoid to myeloid differentiation. Patients with refractory anemia with an excess of blasts responded the most, and did also show a significant decrease in OKIa1-binding cells. Etretinate, on the other hand, did not show any differentiation-inducing activity. The results support earlier reports that retinoic acid might be useful in the treatment of the myelodysplastic syndrome. Whether this in vitro technique offers a possibility to predict the clinical outcome remains to be shown.

Routine use of counterimmunoelectrophoresis for the detection of pneumococcal antigen in sputum.

Sputum samples obtained routinely for culture from patients at a thoracic department were also examined for pneumococcal antigen by means of counterimmunoelectrophoresis (CIE), using a polyvalent antipneumococcal type serum (omniserum). Pneumococci were found in 1.3% of the 880 cultures, whereas pneumococcal antigen was detected with CIE in 6.5%. The validity of these findings was tested by correlating them with the presence of clinical symptoms in those with positive tests and also by antigen detection in ELISA using monoclonal antibodies specific for the C-polysaccharide common to all types of pneumococci. Clinical findings corresponding to confirmed or probable current chest infection were found in 36 of the 48 patients with positive CIE. ELISA was positive in 33 of the 38 patients with positive CIE who were tested. Although the study deals with an unselected material of chest patients, it indicates that CIE is a sensitive method and that it is independent of current antibiotic treatment. Pneumococcal infection is probably of importance in exacerbations of chronic obstructive lung disease, but the clinical usefulness of detecting pneumococcal and other antigens in this patient group needs to be studied further.

Determination of antibodies to pneumococcal C polysaccharide in patients with community-acquired pneumonia.

The pneumococcal C polysaccharide (PnC) is species specific and believed to be a cell wall component of all capsular types. Antibodies against PnC in human sera have been demonstrated previously, but the question of whether a rise in these antibodies occurs during pneumococcal infections has not been investigated. We used an indirect enzyme-linked immunosorbent assay (ELISA) for the estimation of PnC antibodies in 124 hospital-treated patients with pneumonia. In 3 of 6 patients with pneumococcal bacteremia and in 17 of 44 patients with S. pneumoniae isolated in the blood, sputum, or nasopharynx, a significant rise in antibody levels was recorded, accounting for a sensitivity of 38.6%. Of 35 patients with pneumonia of other known or suspected etiology, 1 gave a positive result, corresponding to a specificity of 97.1%. In addition, 3 of 8 patients with PnC antigen in the sputum as the only etiological finding and 5 of 37 patients with unknown etiology gave positive results. The PnC antibodies did not seem to have any protective capacity against pneumonia caused by pneumococci. The ELISA, in which only one antigen preparation was used, was more simple than other tests in which traditional capsular antigen preparations are used. It might therefore be used as a supplemental method in the diagnosis of pneumococcal pneumonia. The problems involved in expressing serum titers obtained with the ELISA are discussed.

Detection of C polysaccharide in Streptococcus pneumoniae in the sputa of pneumonia patients by an enzyme-linked immunosorbent assay.

The pneumococcal C polysaccharide (PnC) is species specific and believed to be a cell wall component of all pneumococcal types. A sandwich enzyme-linked immunosorbent assay (ELISA) for detection of PnC in sputa has been developed by using a monoclonal antiphosphorylcholine antibody and a polyclonal rabbit anti-PnC antiserum in the test system. A 1-year study of adult hospitalized patients with community-acquired pneumonia was performed. A total of 147 patients with clinical and radiological evidence for pneumonia were accepted for the study. Of these, 105 patients provided a sputum sample upon admission to the ward. The sputa were cultured semiquantitatively as well as tested for the presence of antigen. Of the sputum samples from patients with Streptococcus pneumoniae, 27 of 33 (accounting for a sensitivity of 82%) were positive in the ELISA test. Of the sputum samples from patients with pneumonia of some other known or suspected etiology, 32 of 34 (accounting for a specificity of 94%) were negative. In addition, 7 sputum samples from 31 patients with pneumonia of unknown etiology were positive. The ELISA test described here is in our opinion a sensitive and specific test for detecting PnC from S. pneumoniae in sputa from patients with untreated pneumonia.